Numerous studies have reported sex bias in infectious diseases, with bias direction dependent on pathogen and site of infection. is the most common cause of skin and soft tissue infections (SSTIs), yet sex bias in susceptibility to SSTI has not been described. A search of electronic health records revealed an odds ratio of 2.4 for SSTI in males versus females. To investigate the physiological basis of this bias, we compared outcomes between male and female mice in a model of dermonecrosis. Consistent with the epidemiological data, female mice were better protected against SSTI, with reduced dermonecrosis followed later by increased bacterial clearance. Protection in females was disrupted by ovariectomy and restored by short-term estrogen administration. Importantly, this sex bias was mediated by a sex-specific response to the secreted virulence factor α-hemolysin (Hla). Infection with wild-type suppressed inflammatory cytokine production in the skin of female, but not male, mice when compared with infection with an isogenic deletion mutant. This differential response was conserved following injection with Hla alone, demonstrating a direct response to Hla independent of bacterial burden. Additionally, neutrophils, essential for clearing, demonstrated sex-specific bactericidal capacity ex vivo. This work suggests that sex-specific skin innate responsiveness to Hla and neutrophil bactericidal capacity play important roles in limiting SSTI in females. Understanding the molecular mechanisms controlling this sex bias may reveal novel targets to promote host innate defense against skin infection.

Background Prenatal alcohol exposure (PAE) in animal models results in excitatory-inhibitory (E/I) imbalance in neocortex due to alterations in the GABAergic interneuron (IN) differentiation and migration. Thus, E/I imbalance is a potential cause for intellectual disability in individuals with fetal alcohol spectrum disorder (FASD), but whether ethanol (EtOH) changes glutamatergic and GABAergic IN specification during human development remains unknown. Here we created a human cellular model of PAE/FASD and tested the hypothesis that EtOH exposure during differentiation of human pluripotent stem cell-derived neurons (hPSNs) would cause aberrant production of glutamatergic and GABAergic neurons, resulting in E/I imbalance. Methods We applied 50mM EtOH daily to differentiating hPSNs for 50 days to model chronic first trimester exposure. We used quantitative PCR, immunocytochemical, and electrophysiological analysis to examine the effects of EtOH on hPSN specification and functional E/I balance. Results We found that EtOH did not alter neural induction nor general forebrain patterning, and had no effect on the expression of markers of excitatory cortical pyramidal neurons. In contrast, our data revealed highly significant changes to levels of transcripts involved with IN precursor development (e.g. GSX2, DLX1/2/5/6, NR2F2) as well as mature IN specification (e.g. SST, NPY). Interestingly, EtOH did not affect the number of GABAergic neurons generated nor the frequency or amplitude of miniature excitatory and inhibitory postsynaptic currents. Conclusions Similar to in vivo rodent studies, EtOH significantly and specifically altered the expression of genes involved with IN specification from hPSNs but did not cause imbalances of synaptic excitation-inhibition. Thus, our findings corroborate previous studies pointing to aberrant neuronal differentiation as an underlying mechanism of intellectual disability in FASD. However, in contrast to rodent binge models, our chronic exposure model suggests possible compensatory mechanisms that may cause more subtle defects of network processing rather than gross alterations in total E/I balance.

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during in vivo models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in Staphylococcus aureus USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for Staphylococcus species. LAC-p01 was genetically manipulated into an Escherichia coli-S. aureus shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double-and single-stranded (dso and sso) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LACp01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in S. aureus USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in Staphylococcus epidermidis. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of S. aureus systemic infection and successfully complemented an hla mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both in vitro and in vivo experiments are powerful new tools for studies of Staphylococcus. IMPORTANCEPlasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during in vivo studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an S. aureus USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations in vitro and in vivo without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of Staphylococcus species.

Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERβ. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.